Biomarkers Core SUMMARY/ABSTRACT The Biomarkers Core provides a centralized resource for rapid, high throughput quantification of transcripts, proteins, and phosphoproteins. Transcripts will be quantified using quantitative reverse transcriptase PCR (RT-qPCR). In addition, the Biomarkers Core will provide genome-wide microarray analysis using an Illumina Beadstation. Next-generation sequencing is available with Illumina sequencers. Quantification of protein levels in blood or tissue lysates will be done using a MesoScale Imager or by reverse-phase protein array (RPPA). NanoString multi-omics will be available for simultaneous RNA/DNA/protein studies. The Biomarker Core will provide: 1) quantitative mRNA levels for known genes that are involved in proliferation and implicated in cancer progression; 2) identification of unique genes and expression profiles in cells or tissues after a molecular or pharmacologic manipulation; 3) validation of the expression of genes that are initially identified in screening by microarrays or RNA-seq; 4) quantification of protein levels both as an independent validation technique and to determine the relationship between transcript levels and protein levels; 5) assessment of changes in protein/phosphoprotein levels using a comprehensive panel of validated antibodies by RPPA. Specific Aims 1-4 represent interactions between SPORE Projects and the Biomarkers Core. Specific Aim 1 Novel Targeted Strategies for Prevention and Conservative Management of Complex Atypical Hyperplasia and Grade 1 Endometrioid Endometrial Cancer. This project will use RNA-seq following microdissection of glands and stromal cells to identify transcripts in CAH/grade 1 endometrioid endometrial cancer that correspond to progestin-responsive versus progestin-resistant disease, as well as markers of response to everolimus. QPCR and proteomics (NanoString multi-omics or RPPA) will be used for validation. Specific Aim 2. CTNNB1 Mutation and Wnt Pathway Activation Define Clinically Aggressive Endometrioid Endometrial Carcinoma. This project will create at least 7 cell lines containing specific mutations and use transcript and miRNA profiles of the stable lines to identify target genes and pathways. QPCR and proteomic analysis will be used to validate candidate transcripts identified in the screening studies. Specific Aim 3 EphA2 Targeting in Uterine Carcinoma. These studies will evaluate the function of EphA2 in cancer and conduct a clinical trial of EPHARNA, a novel approach to deliver siEphA2 systemically using a neutral liposome nanovehicle. Pre- and post-treatment biopsies from patients enrolled in the trial will be analyzed by RPPA and QPCR. Specific Aim 4 A Framework for Identification of Novel Targeted Therapy Combinations in Endometrial Cancer. Aims 1 and 2 of this proposal will use RPPA analysis (~500 samples) to assess treatment efficacy in clinical trials of PARP and PI3K inhibitors (Aim 1) and to map downstream molecular changes for subsequent design of rational drug combinations (Aim 2). RNA-Seq or NanoString multi-omics can also be used to analyze biopsy specimens.